Worldwide, vaginal transmission is the most prevalent route of infection with HIV. In the SIV/simian AIDS model there is increasing evidence to suggest that SIV in the vaginal lumen is taken up by dendritic cells (DC) and delivered to the draining LN. Moreover, there is evidence for presence of SIV-specific CTL activity in the vaginal mucosa as well as in the draining lymph nodes of rhesus macaques inoculated intra-vaginally with SIV. Thus, a virus-based mucosal gene delivery system that can be taken up locally by DC in the vaginal mucosa and induce local CTL responses is a strong candidate for an HIV vaccine. We recently discovered that our Sindbis virus-based non-replicating particles expressing HIV-1 gag (SIN-gag), which had been successfully used for parenteral immunizations, surprisingly also induced strong local protective CTL responses when administered intravaginally to mice. Alphavirus replicon particles offer the potential to be a safe, non-replicating, mucosal gene delivery vector that can induce mucosal and systemic protective immune responses. We now have developed additional alphavirus-based replicon particles which are chimeric structures combining the replicons of SIN with envelope proteins from Venezuelan equine encephalitis virus (VEE). The mucosal immunogenicity of these novel chimeric particles remains to be investigated. The principal aims of this project are to determine the tropism of alphavirus replicons prepared with SIN and VEE envelope proteins for antigen presenting cells (APC) in the female genital tract of mice and Rhesus macaques and to test whether tropism of replicon particles for DC or other APC contributes to their ability to induce mucosal immune responses in this site. Because the trafficking of the antigen-expressing APC and antigen-specific CTL is important for the dissemination of CTL responses from the vaginal mucosa to local and peripheral lymph nodes, we also will determine the homing properties of the transfected cells as well as the antigen-specific CTL. During year one, we will establish the APC tropism, kinetics of antigen-expression by transfected cells, kinetics of cell trafficking and immune responses in mice. Based on our kinetics studies in mice, during year two in rhesus macaques, we will study the APC-tropism, homing and immunogenicity of the replicons at peak times. Thus, these studies will address several important issues in development of a mucosal HIV vaccine: The tropism of alphavirus-based replicons to APC in the vaginal mucosa, the homing properties of the transfected APC and antigen-specific CTL from the vaginal mucosa to draining local and peripheral lymphoid tissues and how these correlate with the generation of potent local CTL responses.